The efficacy of the analgesic GlyT2 inhibitor, ORG25543, is determined by two connected allosteric sites.

Glycine Transporter 2 (GlyT2) inhibitors have shown considerable potential as analgesics for the treatment of neuropathic pain but also display considerable side effects. One potential source of side effects is irreversible inhibition. In this study, we have characterized the mechanism of ORG25543 inhibition of GlyT2 by first considering three potential ligand binding sites on GlyT2-the substrate site, the vestibule allosteric site and the lipid allosteric site. The three sites were tested using a combination of molecular dynamics simulations and analysis of the inhibition of glycine transport of a series point mutated GlyT2 using electrophysiological methods. We demonstrate that the lipid allosteric site on GlyT2 is the most likely binding site for ORG25543. We also demonstrate that cholesterol derived from the cell membrane can form specific interactions with inhibitor-bound transporters to form an allosteric network of regulatory sites. These observations will guide the future design of GlyT2 inhibitors with the objective of minimising on-target side effects and improving the therapeutic window for the treatment of patients suffering from neuropathic pain.

A non-retinoid antagonist of retinol-binding protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD.

A novel RPE65 inhibitor CU239 suppresses visual cycle and prevents retinal degeneration.

The retinoid visual cycle is an ocular retinoid metabolism specifically dedicated to support vertebrate vision. The visual cycle serves not only to generate light-sensitive visual chromophore 11-cis-retinal, but also to clear toxic byproducts of normal visual cycle (i.e. all-trans-retinal and its condensation products) from the retina, ensuring both the visual function and the retinal health. Unfortunately, various conditions including genetic predisposition, environment and aging may attribute to a functional decline of the all-trans-retinal clearance. To combat all-trans-retinal mediated retinal degeneration, we sought to slow down the retinoid influx from the RPE by inhibiting the visual cycle with a small molecule. The present study describes identification of CU239, a novel non-retinoid inhibitor of RPE65, a key enzyme in the visual cycle. Our data demonstrated that CU239 selectively inhibited isomerase activity of RPE65, with IC50 of 6 µM. Further, our results indicated that CU239 inhibited RPE65 via competition with its substrate all-trans-retinyl ester. Mice with systemic injection of CU239 exhibited delayed chromophore regeneration after light bleach, and conferred a partial protection of the retina against injury from high intensity light. Taken together, CU239 is a potent visual cycle modulator and may have a therapeutic potential for retinal degeneration.

Modulation of NMDA receptor function as a treatment for schizophrenia.

Schizophrenia is a devastating mental illness that afflicts nearly 1% of the world's population. Currently available antipsychotics treat positive symptoms, but are largely ineffective at addressing negative symptoms and cognitive dysfunction. Thus, improved pharmacotherapies that treat all aspects of the disease remain a critical unmet need. There is mounting evidence that links NMDA receptor hypofunction and the expression of schizophrenia, and numerous drug discovery programs have developed agents that directly or indirectly potentiate NMDA receptor-mediated neurotransmission. Several compounds have emerged that show promise for treating all symptom sub-domains in both preclinical models and clinical studies, and we will review recent developments in many of these areas.

Design, synthesis, and SAR of N-((1-(4-(propylsulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1.

The design, synthesis, and structure-activity relationships (SAR) of a series of N-((1-(4-(propylsulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1 (GlyT-1) are described. Optimization of the benzamide and central ring components of the core scaffold led to the identification of a GlyT-1 inhibitor that demonstrated in vivo activity in a rodent cerebral spinal fluid (CSF) glycine model.

Novel Phenylene Lipids That Are Positive Allosteric Modulators of Glycine Receptors and Inhibitors of Glycine Transporter 2.

Chronic pain is a complex condition that remains resistant to current therapeutics. We previously synthesized a series of N-acyl amino acids (NAAAs) that inhibit the glycine transporter, GlyT2, some of which are also positive allosteric modulators of glycine receptors (GlyRs). In this study, we have synthesized a library of NAAAs that contain a phenylene ring within the acyl tail with the objective of improving efficacy at both GlyT2 and GlyRs and also identifying compounds that are efficacious as dual-acting modulators to enhance glycine neurotransmission. The most efficacious positive allosteric modulator of GlyRs was 2-[8-(2-octylphenyl)octanoylamino]acetic acid (8-8 OPGly) which potentiates the EC5 for glycine activation of GlyRα1 by 1500% with an EC50 of 664 nM. Phenylene-containing NAAAs with a lysine headgroup were the most potent inhibitors of GlyT2 with (2S)-6-amino-2-[8-(3-octylphenyl)octanoylamino]hexanoic acid (8-8 MPLys) inhibiting GlyT2 with an IC50 of 32 nM. The optimal modulator across both proteins was (2S)-6-amino-2-[8-(2-octylphenyl)octanoylamino]hexanoic acid (8-8 OPLys), which inhibits GlyT2 with an IC50 of 192 nM and potentiates GlyRs by up to 335% at 1 µM. When tested in a dual GlyT2/GlyRα1 expression system, 8-8 OPLys caused the greatest reductions in the EC50 for glycine. This suggests that the synergistic effects of a dual-acting modulator cause greater enhancements in glycinergic activity compared to single-target modulators and may provide an alternate approach to the development of new non-opioid analgesics for the treatment of chronic pain.

Structural similarities between SARS-CoV2 3CLpro and other viral proteases suggest potential lead molecules for developing broad spectrum antivirals.

Considering the significant impact of the recent COVID-19 outbreak, development of broad-spectrum antivirals is a high priority goal to prevent future global pandemics. Antiviral development processes generally emphasize targeting a specific protein from a particular virus. However, some antiviral agents developed for specific viral protein targets may exhibit broad spectrum antiviral activity, or at least provide useful lead molecules for broad spectrum drug development. There is significant potential for repurposing a wide range of existing viral protease inhibitors to inhibit the SARS-CoV2 3C-like protease (3CLpro). If effective even as relatively weak inhibitors of 3CLpro, these molecules can provide a diverse and novel set of scaffolds for new drug discovery campaigns. In this study, we compared the sequence- and structure-based similarity of SARS-CoV2 3CLpro with proteases from other viruses, and identified 22 proteases with similar active-site structures. This structural similarity, characterized by secondary-structure topology diagrams, is evolutionarily divergent within taxonomically related viruses, but appears to result from evolutionary convergence of protease enzymes between virus families. Inhibitors of these proteases that are structurally similar to the SARS-CoV2 3CLpro protease were identified and assessed as potential inhibitors of SARS-CoV2 3CLpro protease by virtual docking. Several of these molecules have docking scores that are significantly better than known SARS-CoV2 3CLpro inhibitors, suggesting that these molecules are also potential inhibitors of the SARS-CoV2 3CLpro protease. Some have been previously reported to inhibit SARS-CoV2 3CLpro. The results also suggest that established inhibitors of SARS-CoV2 3CLpro may be considered as potential inhibitors of other viral 3C-like proteases.

The novel visual cycle inhibitor (±)-RPE65-61 protects retinal photoreceptors from light-induced degeneration.

The visual cycle refers to a series of biochemical reactions of retinoids in ocular tissues and supports the vision in vertebrates. The visual cycle regenerates visual pigments chromophore, 11-cis-retinal, and eliminates its toxic byproducts from the retina, supporting visual function and retinal neuron survival. Unfortunately, during the visual cycle, when 11-cis-retinal is being regenerated in the retina, toxic byproducts, such as all-trans-retinal and bis-retinoid is N-retinylidene-N-retinylethanolamine (A2E), are produced, which are proposed to contribute to the pathogenesis of the dry form of age-related macular degeneration (AMD). The primary biochemical defect in Stargardt disease (STGD1) is the accelerated synthesis of cytotoxic lipofuscin bisretinoids, such as A2E, in the retinal pigment epithelium (RPE) due to mutations in the ABCA4 gene. To prevent all-trans-retinal-and bisretinoid-mediated retinal degeneration, slowing down the retinoid flow by modulating the visual cycle with a small molecule has been proposed as a therapeutic strategy. The present study describes RPE65-61, a novel, non-retinoid compound, as an inhibitor of RPE65 (a key enzyme in the visual cycle), intended to modulate the excessive activity of the visual cycle to protect the retina from harm degenerative diseases. Our data demonstrated that (±)-RPE65-61 selectively inhibited retinoid isomerase activity of RPE65, with an IC50 of 80 nM. Furthermore, (±)-RPE65-61 inhibited RPE65 via an uncompetitive mechanism. Systemic administration of (±)-RPE65-61 in mice resulted in slower chromophore regeneration after light bleach, confirming in vivo target engagement and visual cycle modulation. Concomitant protection of the mouse retina from high-intensity light damage was also observed. Furthermore, RPE65-61 down-regulated the cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway, decreased the inflammatory factor, and attenuated retinal apoptosis caused by light-induced retinal damage (LIRD), which led to the preservation of the retinal function. Taken together, (±)-RPE65-61 is a potent visual cycle modulator that may provide a neuroprotective therapeutic benefit for patients with STGD and AMD.

Novel serotonin type 3 receptor partial agonists for the potential treatment of irritable bowel syndrome.

Serotonin type 3 (5-HT(3)) receptor partial agonists are being targeted as potential new drugs for the treatment of irritable bowel syndrome (IBS). Two new chemical series bearing indazole and indole cores have exhibited nanomolar binding affinity for the h5-HT(3)A receptor. A range of partial agonist activities in HEK cells heterologously expressing the h5-HT(3)A receptor were measured for the indazole series. Excellent 5-HT(3) receptor selectivity, favorable in vitro metabolic stability and CYP inhibition properties, and good oral in vivo potency in the murine von Bezold-Jarisch reflex model is exemplified thereby indicating the series to have potential utility as improved IBS agents.

Inhibition of Glycine Re-Uptake: A Potential Approach for Treating Pain by Augmenting Glycine-Mediated Spinal Neurotransmission and Blunting Central Nociceptive Signaling.

Among the myriad of cellular and molecular processes identified as contributing to pathological pain, disinhibition of spinal cord nociceptive signaling to higher cortical centers plays a critical role. Importantly, evidence suggests that impaired glycinergic neurotransmission develops in the dorsal horn of the spinal cord in inflammatory and neuropathic pain models and is a key maladaptive mechanism causing mechanical hyperalgesia and allodynia. Thus, it has been hypothesized that pharmacological agents capable of augmenting glycinergic tone within the dorsal horn may be able to blunt or block aberrant nociceptor signaling to the brain and serve as a novel class of analgesics for various pathological pain states. Indeed, drugs that enhance dysfunctional glycinergic transmission, and in particular inhibitors of the glycine transporters (GlyT1 and GlyT2), are generating widespread interest as a potential class of novel analgesics. The GlyTs are Na+/Cl--dependent transporters of the solute carrier 6 (SLC6) family and it has been proposed that the inhibition of them presents a possible mechanism by which to increase spinal extracellular glycine concentrations and enhance GlyR-mediated inhibitory neurotransmission in the dorsal horn. Various inhibitors of both GlyT1 and GlyT2 have demonstrated broad analgesic efficacy in several preclinical models of acute and chronic pain, providing promise for the approach to deliver a first-in-class non-opioid analgesic with a mechanism of action differentiated from current standard of care. This review will highlight the therapeutic potential of GlyT inhibitors as a novel class of analgesics, present recent advances reported for the field, and discuss the key challenges associated with the development of a GlyT inhibitor into a safe and effective agent to treat pain.

Identification of Transthyretin Tetramer Kinetic Stabilizers That Are Capable of Inhibiting the Retinol-Dependent Retinol Binding Protein 4-Transthyretin Interaction: Potential Novel Therapeutics for Macular Degeneration, Transthyretin Amyloidosis, and Their Common Age-Related Comorbidities.

Dissociation of transthyretin (TTR) tetramers may lead to misfolding and aggregation of proamyloidogenic monomers, which underlies TTR amyloidosis (ATTR) pathophysiology. ATTR is a progressive disease resulting from the deposition of toxic fibrils in tissues that predominantly presents clinically as amyloid cardiomyopathy and peripheral polyneuropathy. Ligands that bind to and kinetically stabilize TTR tetramers prohibit their dissociation and may prevent ATTR onset. Drawing from clinically investigated AG10, we designed a constrained congener (14) that exhibits excellent TTR tetramer binding potency, prevents TTR aggregation in a gel-based assay, and possesses desirable pharmacokinetics in mice. Additionally, 14 significantly lowers murine serum retinol binding protein 4 (RBP4) levels despite a lack of binding at that protein's all-trans-retinol site. We hypothesize that kinetic stabilization of TTR tetramers via 14 is allosterically hindering all-trans-retinol-dependent RBP4-TTR tertiary complex formation and that the compound could present ancillary therapeutic utility for indications treated with RBP4 antagonists, such as macular degeneration.

Synthesis of 2-aminobenzoxazoles using tetramethyl orthocarbonate or 1,1-dichlorodiphenoxymethane.

The synthesis of 2-aminobenzoxazoles can be readily achieved by two versatile, one-pot procedures utilizing commercially available tetramethyl orthocarbonate or 1,1-dichlorodiphenoxymethane, an amine, and an optionally substituted 2-aminophenol. The reactions were conducted under mild conditions and provided 2-aminobenzoxazoles in modest to excellent yields. A variety of amines and substituted 2-aminophenols were used to investigate the scope of the reactions.

Discovery of Bispecific Antagonists of Retinol Binding Protein 4 That Stabilize Transthyretin Tetramers: Scaffolding Hopping, Optimization, and Preclinical Pharmacological Evaluation as a Potential Therapy for Two Common Age-Related Comorbidities.

Accumulation of cytotoxic lipofuscin bisretinoids may contribute to atrophic age-related macular degeneration (AMD) pathogenesis. Retinal bisretinoid synthesis depends on the influx of serum all-trans-retinol (1) delivered via a tertiary retinol binding protein 4 (RBP4)-transthyretin (TTR)-retinol complex. We previously identified selective RBP4 antagonists that dissociate circulating RBP4-TTR-retinol complexes, reduce serum RBP4 levels, and inhibit bisretinoid synthesis in models of enhanced retinal lipofuscinogenesis. However, the release of TTR by selective RBP4 antagonists may be associated with TTR tetramer destabilization and, potentially, TTR amyloid formation. We describe herein the identification of bispecific RBP4 antagonist-TTR tetramer kinetic stabilizers. Standout analogue (±)-44 possesses suitable potency for both targets, significantly lowers mouse plasma RBP4 levels, and prevents TTR aggregation in a gel-based assay. This new class of bispecific compounds may be especially important as a therapy for dry AMD patients who have another common age-related comorbidity, senile systemic amyloidosis, a nongenetic disease associated with wild-type TTR misfolding.

Photoswitchable ORG25543 Congener Enables Optical Control of Glycine Transporter 2.

Glycine neurotransmission in the dorsal horn of the spinal cord plays a key role in regulating nociceptive signaling, but in chronic pain states reduced glycine neurotransmission is associated with the development of allodynia and hypersensitivity to painful stimuli. This suggests that restoration of glycine neurotransmission may be therapeutic for the treatment of chronic pain. Glycine transporter 2 inhibitors have been demonstrated to enhance glycine neurotransmission and provide relief from allodynia in rodent models of chronic pain. In recent years, photoswitchable compounds have been developed to provide the possibility of controlling the activity of target proteins using light. In this study we have developed a photoswitchable noncompetitive inhibitor of glycine transporter 2 that has different affinities for the transporter at 365 nm compared to 470 nm light.

Stringent response governs the oxidative stress resistance and virulence of Francisella tularensis.

Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and, based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. This work is an important step forward towards understanding the complex regulatory network underlying the oxidative stress response of F. tularensis.

Design, Synthesis, and Preclinical Efficacy of Novel Nonretinoid Antagonists of Retinol-Binding Protein 4 in the Mouse Model of Hepatic Steatosis.

Retinol-binding protein 4 (RBP4) serves as a transporter for all- trans-retinol (1) in the blood, and it has been proposed to act as an adipokine. Elevated plasma levels of the protein have been linked to diabetes, obesity, cardiovascular diseases, and nonalcoholic fatty liver disease (NAFLD). Recently, adipocyte-specific overexpression of RBP4 was reported to cause hepatic steatosis in mice. We previously identified an orally bioavailable RBP4 antagonist that significantly lowered RBP4 serum levels in Abca4-/- knockout mice with concomitant normalization of complement system protein expression and reduction of bisretinoid formation within the retinal pigment epithelium. We describe herein the discovery of novel RBP4 antagonists 48 and 59, which reduce serum RBP4 levels by >80% in mice upon acute oral dosing. Furthermore, 59 demonstrated efficacy in the transgenic adi-hRBP4 murine model of hepatic steatosis, suggesting that RBP4 antagonists may also have therapeutic utility for the treatment of NAFLD.

Modulation of Glycine-Mediated Spinal Neurotransmission for the Treatment of Chronic Pain.

Chronic pain constitutes a significant and expanding worldwide health crisis. Currently available analgesics poorly serve individuals suffering from chronic pain, and new therapeutic agents that are more effective, safer, and devoid of abuse liabilities are desperately needed. Among the myriad of cellular and molecular processes contributing to chronic pain, spinal disinhibition of pain signaling to higher cortical centers plays a critical role. Accumulating evidence shows that glycinergic inhibitory neurotransmission in the spinal cord dorsal horn gates nociceptive signaling, is essential in maintaining physiological pain sensitivity, and is diminished in pathological pain states. Thus, it is hypothesized that agents capable of enhancing glycinergic tone within the dorsal horn could obtund nociceptor signaling to the brain and serve as analgesics for persistent pain. This Perspective highlights the potential that pharmacotherapies capable of increasing inhibitory spinal glycinergic neurotransmission hold in providing new and transformative analgesic therapies for the treatment of chronic pain.

Glycine transporter-1 inhibitors: a patent review (2011-2016).

INTRODUCTION: Numerous research groups have developed GlyT-1 inhibitors in the pursuit of providing a novel antipsychotic treatment for schizophrenia. Despite multiple compounds advancing into clinical trials, a GlyT-1 inhibitor has yet to emerge to treat patients. However, the approach remains heavily investigated as it presents potential therapeutic utility for several other CNS and non-CNS-related indications. AREAS COVERED: This review discusses various GlyT-1 inhibitor chemotypes identified and provides an overview of patent applications filed and published during the period of 2011-2016. The review largely focuses on composition of matter patent applications, although two recently disclosed method of use patents are discussed. Clinical reports are also disseminated. EXPERT OPINION: Mounting clinical failures with schizophrenic patients have blunted enthusiasm for GlyT-1 inhibition as an approach to treat the disease. However, research in the area remains quite active, as therapeutic potential for several additional indications has emerged. There are numerous and diverse GlyT-1 chemotypes now available that exhibit differentiating modes of binding and ligand-target binding kinetics, and this rich diversity of chemical matter may help further elucidate the target's pharmacological role in various indications and lead to the identification of a compound with optimal properties that may someday become a drug.

Inhibitors of Glycine Transporter-1: Potential Therapeutics for the Treatment of CNS Disorders.

Glycine acts as an inhibitory neurotransmitter at glycine receptor (GlyR)-enriched synapses and as an obligatory co-agonist at the N-methyl-D-aspartate (NMDA) receptor, where it facilitates neuronal excitation. Two high-affinity and substrate selective transporters, glycine transporter-1 and glycine transporter-2 (GlyT-1 and GlyT-2), regulate extracellular glycine concentrations within the CNS and as such, play critical roles in maintaining a balance between inhibitory and excitatory neurotransmission. GlyT-1 inhibition has been extensively examined as a potential means by which to treat several CNS disorders that include schizophrenia, depression, anxiety, obsessive compulsive disorder (OCD), and addiction. More recently, preclinical studies have emerged that indicate the approach may also promote neuroprotection, provide a pharmacotherapeutic strategy for autism spectrum disorders (ASDs), and treat symptomology associated with pain, Parkinson's disease, and epilepsy. This review examines the pharmacological aspects of GlyT-1 inhibition and describes drug discovery and development efforts toward the identification of novel inhibitors.

Synthesis and Biological Evaluation of N-((1-(4-(Sulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide Inhibitors of Glycine Transporter-1.

We previously disclosed the discovery of rationally designed N-((1-(4-(propylsulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1 (GlyT-1), represented by analogues 10 and 11. We describe herein further structure-activity relationship exploration of this series via an optimization strategy that primarily focused on the sulfonamide and benzamide appendages of the scaffold. These efforts led to the identification of advanced leads possessing a desirable balance of excellent in vitro GlyT-1 potency and selectivity, favorable ADME and in vitro pharmacological profiles, and suitable pharmacokinetic and safety characteristics. Representative analogue (+)-67 exhibited robust in vivo activity in the cerebral spinal fluid glycine biomarker model in both rodents and nonhuman primates. Furthermore, rodent microdialysis experiments also demonstrated that oral administration of (+)-67 significantly elevated extracellular glycine levels within the medial prefrontal cortex (mPFC).